Exosomes derived from MSCs ameliorate retinal laser injury partially by inhibition of MCP-1

Bo Yu, Hui Shao, Chang Su, Yuanfeng Jiang, Xiteng Chen, Lingling Bai, Yan Zhang, Qiutang Li, Xiaomin Zhang, Xiaorong Li, Bo Yu, Hui Shao, Chang Su, Yuanfeng Jiang, Xiteng Chen, Lingling Bai, Yan Zhang, Qiutang Li, Xiaomin Zhang, Xiaorong Li

Abstract

Although accumulated evidence supports the notion that mesenchymal stem cells (MSCs) act in a paracrine manner, the mechanisms are still not fully understood. Recently, MSC-derived exosomes (MSC-Exos), a type of microvesicle released from MSCs, were thought to carry functional proteins and RNAs to recipient cells and play therapeutic roles. In the present study, we intravitreally injected MSCs derived from either mouse adipose tissue or human umbilical cord, and their exosomes to observe and compare their functions in a mouse model of laser-induced retinal injury. We found that both MSCs and their exosomes reduced damage, inhibited apoptosis, and suppressed inflammatory responses to obtain better visual function to nearly the same extent in vivo. Obvious down-regulation of monocyte chemotactic protein (MCP)-1 in the retina was found after MSC-Exos injection. In vitro, MSC-Exos also down-regulated MCP-1 mRNA expression in primarily cultured retinal cells after thermal injury. It was further demonstrated that intravitreal injection of an MCP-1-neutralizing antibody promoted the recovery of retinal laser injury, whereas the therapeutic effect of exosomes was abolished when MSC-Exos and MCP-1 were administrated simultaneously. Collectively, these results suggest that MSC-Exos ameliorate laser-induced retinal injury partially through down-regulation of MCP-1.

Figures

Figure 1
Figure 1
Micrographs of scanning electron microscopy of (A) maMSC-Exos, (B) hucMSC-Exos and (C) f-Exos show spheroid shaped vesicles at the diameter of about 40–100 nm. Scale bar = 200 nm.
Figure 2. MaMSCs and maMSC-Exos promote the…
Figure 2. MaMSCs and maMSC-Exos promote the recovery of laser-induced retinal injury.
(A) Micrographs of retinal histological images (H&E staining) before injury (A1) and 3 days after injury (A2). Main histological parameters are presented with lines. Scale bar = 50 μm. (B) Effect of maMSC-Exos at different concentrations on diameter of retinal disordered area (B1) and ONL defect area (B2) at 3rd day post-injury. (C) Lesion areas after treatment with PBS, maMSCs or maMSC-Exos on 1st, 3rd, 7th and 14th days post-injury. n > 6, *p < 0.05. (D1) Amplitudes of dark-adapted ERG at 3, 14 and 60 days post-injury of PBS, maMSC- or maMSC-Exo-treated group. A wave and b wave amplitudes of dark adapted ERG (D2,D3) and light-adapted ERG (D4,D5) at 3, 7, 14, 21, 40 and 60 days post-injury were analysed and compared among the three groups. n = 7, *p < 0.05 (E) TUNEL staining of injury sections treated with PBS, maMSCs or maMSC-Exos at 1, 3, 7 and 14 days post-injury. n = 8, *p < 0.05. (F) Diameter of retinal disordered areas (F1), diameter of ONL defect areas (F2) and ERG responses (F3) at 3 days post-injury of maMSC-Exo- and f-Exo-treated groups. n = 6, *p < 0.05.
Figure 3. Representative examples of H&E staining…
Figure 3. Representative examples of H&E staining images at different time points of evaluation.
One day after injury of PBS (A1), maMSC-Exo (B1) or maMSC (C1) treated group. Three days after injury of PBS (A2), maMSC-Exo (B2) or maMSC (C2) treated group. Seven days after injury of PBS (A3), maMSC-Exo (B3) or maMSC (C3) treated group. Fourteen days after injury of PBS (A4), maMSC-Exo (B4) or maMSC (C4) treated group. Scale bar = 50 μm.
Figure 4. Representative examples of TUNEL staining…
Figure 4. Representative examples of TUNEL staining images at different time points of evaluation.
One day after injury of PBS(A1), maMSC-Exo (B1) or maMSC (C1) treated group. Three days after injury of PBS (A2), maMSC-Exo (B2) or maMSC (C2) treated group. Seven days after injury of PBS (A3), maMSC-Exo (B3) or maMSC (C3) treated group. Fourteen days after injury of PBS(A4), maMSC-Exo (B4) or maMSC (C4) treated group.(Scale bar = 20 μm; green: TUNEL staining of apoptotic cells; blue: DAPI staining of nucleus).
Figure 5. HucMSCs and hucMSC-Exos help protect…
Figure 5. HucMSCs and hucMSC-Exos help protect retina in mouse retinal laser injury model.
(A) Diameter of retinal disordered areas (A1) and ONL defect areas (A2) after treatment with PBS, hucMSCs or hucMSC-Exos at 1, 3, 7 and 14 days post-injury. n > 6, *p < 0.05. (B) Number of TUNEL positive cells/section of PBS-, hucMSC- and hucMSC-Exo-treated groups on 1st, 3rd, 7th and 14th days post-injury. n = 6, *p < 0.05. (C) A wave and b wave amplitudes of dark adapted ERG (C1,C2) and light-adapted ERG (C3,C5) at 3, 7, 14, 21, 40 and 60 days post-injury were analysed and compared among the three groups. n = 7, *p < 0.05.
Figure 6. MaMSC-Exos suppress the injury-induced inflammation…
Figure 6. MaMSC-Exos suppress the injury-induced inflammation in vivo and in vitro.
(A1–A3) Relative mRNA expression of MCP-1, TNF-α and ICAM1 of PBS- or MaMSC-Exo-treated group at 1, 3, 7 and 14 days post-injury. n = 7, *p B) Immunohistochemistry staining images of PBS- or MaMSC-Exo-treated group at 3 days post-injury. (C1,C2) Western blotting analyses of MCP-1 in retina/RPE/Choroid tissues from PBS- or MaMSC-Exo-treated group at 1, 3, 7 and 14 days post-injury. n = 3, *p < 0.05. (D1) Cell number of normal, thermo injury or MaMSC-Exo-treated group at 3 days after thermo injury. (D2) Relative mRNA expression of MCP-1 in normal, thermo injury or MaMSC-Exo-treated group at 3 days after thermo injury.
Figure 7. Representative examples of immunohistochemistry staining…
Figure 7. Representative examples of immunohistochemistry staining images at different time points of evaluation.
One day after injury of PBS (A1) or maMSC-Exo (B1) treated group. Seven days after injury of PBS (A1) or maMSC-Exo (B1) treated group. Fourteen days after injury of PBS (A1) or maMSC-Exo (B1) treated group. Scale bar = 20 μm.
Figure 8. Effect of MCP-1 regulation on…
Figure 8. Effect of MCP-1 regulation on laser induced retinal injury in mice.
(A1) Representative H&E staining images of PBS- or MCP-1 neutralizing antibody-treated groups at 3 days post-injury. Scale bar = 50 μm. Diameter of retinal disordered areas (A2) and diameter of ONL defect areas (A3) of PBS- or MCP-1 neutralizing antibody-treated group at 3 days post-injury. n = 6, *p 
All figures (8)

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